The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans and Category A select agents. There are seven serotypes of the BoNTs (termed A-G). The current penta-serotype vaccine is in limited supply and there are no approved therapies against botulism. During studies on BoNT entry into neurons, the B subunit of cholera toxin (CTB) was observed to enter BoNT-intoxicated neurons with the same efficiency as untreated cells. Since cholera toxin has been used to deliver heterologous proteins into cells, CTB or a related B subunit of the heat-labile enterotoxins of Escherichia coli (LTB) wil be developed as a platform to deliver therapies into BoNT-intoxicated neurons. The R21 component is a proof of concept to engineer a B subunit-based platform to deliver a heterologous cargo into the cytosol of a BoNT- intoxicated neuron. There are three aims: identify the most efficient B subunit for entry into BoNT-intoxicated neurons, engineer a cargo-A2 linker to assemble onto the B subunit delivery platform, and determine if the B subunit platform can deliver cargo into a BoNT-intoxicated neuron. The R33 component will optimize the B subunit as a therapy. There are 3 aims: optimize entry of the B subunit platform into neurons, optimize the cargo-A2 linker for assembly onto the B subunit platform, and deliver a pan-serotype therapy into a BoNT-intoxicated neuron and test this therapy in BoNT-intoxicated mice. Completion of this proposal will provide a platform for therapies against botulism that may also be useful as a delivery platform to treat other infectious neurological diseases and diseases of the nervous system. PUBLIC HEALTH RELEVANCE: The botulinum neurotoxins (BoNTs) are the most toxic proteins for humans. The determination that the B subunit of cholera toxin enters BoNT-intoxicated neurons provides a basis to develop a platform to deliver therapies to treat BoNT-intoxicated cells to reverse the paralysis associated with botulism. This platform may also have utility to deliver therapies against other neurological diseases.